Functional feline pancreatic cells from adipose tissue

ABSTRACT

Compositions and methods are described herein for transdifferentiation of multipotent stromal cells into cells that can express insulin.

PRIORITY

This application claims benefit of the priority filing date of U.S.Provisional Patent Application Ser. No. 62/537,712, filed Jul. 27, 2017,the contents of which are specifically incorporated by reference hereinin their entirety.

BACKGROUND

Diabetes mellitus is one of the most prevalent feline endocrinopathies.About 15-20% of feline diabetes cases are caused by decreased β-cellnumbers or insulin resistance (Rand et al., J Nutrition 134:1072S-1080S(2004); Sittinger et al. Curr. Opin. Biotech. 15:411-418 (2004)). Thecondition affects all breeds and sexes and is associated withreproductive sterilization^(5,6), obesity, physical inactivity⁷, andadvancing age⁸ and drug side effects (McCann et al., J. Feline Med &Surg 9: 289-299 (2007); Pancciera et al., J Am Vet Med Assoc 197:1504-1508 (1990); Prahl et al., J. Feline Med & Surg 9: 351-358 (2007);O'Brien, Molec Cell Endocrinol 197: 213-219 (2002)). To date, no singlecause or effective cure has been identified.

Serious complications associated with unregulated glucose levels includeincreased bone fractures, cardiovascular disease, and neurologicaldysfunction, among others (Schwartz Calcified Tissue Internat 73:515-519(2003); Shehadeh & Regan, Clin. Cardiol. 18: 301-305 (1995); Mizisin etal., J Neuropath & Exp. Neurol. 61-872-884 (2002)). Contemporarytreatment consists of diet and weight management with exogenous insulinadministration to replace that normally produced by pancreatic β cells(Fu et al., Curr. Diabetes Rev. 9: 25 (2013)). Available insulinformulations do not share the feline amino acid sequence. Though insulinmaintains biological activity across species, sequence differences mayaffect activity and stimulate the immune system (Betsholtz et al.,Diabetes 39: 118-122 (1990); Chance et al., Science 161: 165-167(1968)). Additionally, insulin administration is typically customizedfor individual patients, a challenging and time-consuming process. Thetime and costs associated with establishing and revising insulin therapyand daily injections can be a burden on the owners.

SUMMARY

Methods and compositions are described herein for transdifferentiationof feline starting cells such as adipose-derived multipotent stromalcells (ASCs) to generate pancreatic β cells. Such starting cells (e.g.,ASCs) can be obtained from adipose tissues, for example, fromsubcutaneous adipose tissue or from reproductive organs (e.g., when theyare removed during routine feline sterilization procedures). Results aredescribed herein that quantify the transdifferentiation capability offeline starting cells such as ASCs. The feline starting cells culturedin pancreatic β cell induction medium had better insulin production andglucose response compared to that cultured in stromal medium.

One method described to generate pancreatic β cells can include:

-   -   a. Stage 1: culturing adult adipose-derived multipotent stromal        (stem) cells (ASCs) for about 1-4 days in a first culture medium        comprising insulin-transferrin-selenium, activin A, sodium        butyrate, and 2-mercapethanol to generate a first population of        cells that express at least one of the following genes: Nkx 6.1,        Pax6, Isl1, or Glut-2;    -   b. Stage 2: culturing the first population of cells for 4 to 8        days in a second culture medium comprising        insulin-transferrin-selenium, and taurine to generate a second        population of cells; and    -   c. Stage 3: culturing the second population of cells for 2 to 6        days in a third culture medium comprising        insulin-transferrin-selenium, taurine, glucagon-like peptide 1        (GLP-1), nicotinamide, pentagastrin, and betacellulin to        generate a third population of cells.        Cells in the second population typically express Nkx 6.1, Pax6,        Isl1, and/or Glut-2 at greater expression levels than cells in        the first population. Cells in the third population also        typically express Nkx 6.1, Pax6, Isl1, and/or Glut-2 at greater        expression levels than cells in the second population.

The methods can also include administering the third population of cells(or a portion thereof) to a subject, for example, a feline subject.

In some cases, the first culture medium, the second culture medium, andthe third culture medium can further include at least one, at least two,at least three, or at least four of the following: basic fibroblastgrowth factor (bFGF), hepatocyte growth factor (HGF), laminin, N-2supplement, or B-27 supplement.

In some cases the first culture medium, the second culture medium, andthe third culture medium can also contain glucose, and/or serum albumin(e.g., bovine serum albumin).

Cells in the second population of cells and/or the third population ofcells can express insulin. However, cells in the first population ofcells typically do not express detectable amounts of insulin mRNA asdetected by quantitative polymerase chain polymerase reaction.

Compositions are also described herein.

For example, one type of cell composition can include a first populationof cells made by: culturing adult adipose-derived multipotent stromal(stem) cells (ASCs) from feline adipose tissue for about 1-4 days in afirst culture medium comprising insulin-transferrin-selenium, activin A,sodium butyrate, and 2-mercapethanol to generate a first population ofcells that express at least one of the following genes: Nkx 6.1, Pax6,Isl1, or Glut-2.

Another type of cell composition can include a second population ofcells made by:

-   -   a. Stage 1: culturing adult adipose-derived multipotent stromal        (stem) cells (ASCs) for about 1-4 days in a first culture medium        comprising insulin-transferrin-selenium, activin A, sodium        butyrate, and 2-mercapethanol to generate a first population of        cells that express at least one of the following genes: Nkx 6.1,        Pax6, Isl1, or Glut-2; and    -   b. Stage 2: culturing the first population of cells for 4 to 8        days in a second culture medium comprising        insulin-transferrin-selenium, and taurine to generate a second        population of cells.

Another type of cell composition can include a third population of cellsmade by:

-   -   a. Stage 1: culturing adult adipose-derived multipotent stromal        (stem) cells (ASCs) for about 1-4 days in a first culture medium        comprising insulin-transferrin-selenium, activin A, sodium        butyrate, and 2-mercapethanol to generate a first population of        cells that express at least one of the following genes: Nkx 6.1,        Pax6, Isl1, or Glut-2;    -   b. Stage 2: culturing the first population of cells for 4 to 8        days in a second culture medium comprising        insulin-transferrin-selenium, and taurine to generate a second        population of cells; and    -   c. Stage 3: culturing the second population of cells for 2 to 6        days in a third culture medium comprising        insulin-transferrin-selenium, taurine, glucagon-like peptide 1        (GLP-1), nicotinamide, pentagastrin, and betacellulin to        generate a third population of cells.

Composition are also included herein that are useful fortransdifferentiation of cells. Such compositions can be formulated as aculture medium, or as a dry composition or a concentrated liquid that islater rehydrated or diluted, respectively, to generate the culturemedium.

For example, one type of composition can include at least four, or atleast five, or at least six, or at least seven, or at least eight, or atleast nine, or at least ten of the following: glucose, bovine serumalbumin, insulin-transferrin-selenium, activin A, sodium butyrate,2-mercapethanol, N-2 supplement, B-27 supplement, laminin, hepatocytegrowth factor (HGF), or basic fibroblast growth factor (bFGF).

Another type of composition can include at least four, or at least five,or at least six, or at least seven, or at least eight of the following:glucose, bovine serum albumin, insulin-transferrin-selenium, taurine,laminin, basic fibroblast growth factor (bFGF), N-2 supplement, B-27supplement, or human hepatocyte growth factor (HGF).

Another type of composition can include at least four, or at least five,or at least six, or at least seven, or at least eight, or at least nine,or at least ten, or at least eleven, or at least twelve of thefollowing: glucose, bovine serum albumin, insulin-transferrin-selenium,taurine, glucagon-like peptide 1 (GLP-1), nicotinamide, non-essentialamino acids, pentagastrin, N-2 supplement, B-27 supplement, humanhepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF),laminin, or betacellulin.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are photomicrographs of feline adipose-derived multipotentstromal (stem) cells (ASCs) following culture in stromal medium. FIG. 1Ais a photomicrograph showing ASCs after culturing in stromal medium. Thescale is a 5× scale, and the scale bar=500 μm. FIG. 1B is aphotomicrograph showing alizarin red stained ASCs after culturing inosteogenic medium. The scale is a 5×scale, and the scale bar=500 μm.Alizarin red staining illustrates calcium deposition. FIG. 1C is aphotomicrograph showing oil red O lipid stained ASCs after culture inAdipogenic medium. The scale is a 40×scale, and the scale bar=50 μm.

FIG. 2A-2C are light photomicrographs of fresh feline P3 ASCs. FIG. 2Ashows a light photomicrograph of fresh male feline P3 ASCs cultured instromal medium. FIG. 2B shows a light photomicrograph of fresh malefeline P3 ASCs cultured in pancreatic β-cell induction medium andtransferred to the normal culture plate. FIG. 2C shows a lightphotomicrograph of fresh female feline P3 ASCs cultured in pancreaticβ-cell induction medium and transferred to the normal culture plate. 5×magnification. Scale bar=500 μm.

FIGS. 3A-3C are light photomicrographs of dithizone-stained fresh felineP3 ASCs. FIG. 3A shows a light photomicrograph of fresh male feline P3ASCs stained with dithizone cultured in stromal medium. FIG. 3B shows alight photomicrograph of fresh male feline P3 dithizone-stained ASCscultured in pancreatic β-cell induction medium. FIG. 3C shows a lightphotomicrograph of fresh female feline P3 dithizone-stained ASCscultured in pancreatic β-cell induction medium. Dithizone binds zincions present in the islet's beta cells, and therefore stains the islets.20× magnification, scale bar=100 μm (A, B); 40× magnification, scalebar=50 μm.

FIG. 4 illustrates fluorescent photomicrographs of cells cultured instromal (top three panels) or induction medium (bottom three panels) andlabeled with antibodies against insulin (middle two panels, red in theoriginal) and actin (left two panels, blue in original). As illustrated,cells cultured in stromal media do not express insulin, but cellsincubated in induction medium do express insulin.

FIG. 5 graphically illustrates results of a glucose challenge assay ofcells generated by the methods described herein. Insulin secretion fromdifferentiated islet-like cell clusters exposed to distinct glucoseconcentrations in the medium. Columns with distinct superscripts aresignificantly different among differentiation stages within gender.Insulin secretion from differentiated islet-like cell clusters atstimulated concentrations of (25 and 55 mM) glucose are shown.

FIG. 6A-6B illustrate transmission electron photomicrographs of felineASCs. FIG. 6A illustrates a transmission electron photomicrograph offeline ASCs cultured in stromal medium. FIG. 6B illustrates atransmission electron photomicrograph of feline ASCs cultured ininduction medium. Legend: M—mitochondria, G—secretory granules. Scalebar=2 μm.

FIG. 7A-7B show transmission electron photomicrographs demonstratinginsulin labeled with anti-insulin antibodies (arrows) in feline ASCscultured in β pancreatic cell medium. FIG. 7A shows transmissionelectron photomicrographs of feline ASCs cultured in stromal medium.FIG. 7B shows transmission electron photomicrographs of feline ASCscultured in β cell induction medium. Scale bar=0.5 μm.

FIG. 8A-8B show scanning electron photomicrographs of feline ASCs. FIG.8A shows scanning electron photomicrographs of feline ASCs cultured instromal medium. FIG. 8B shows scanning electron photomicrographs offeline ASCs cultured in β cell induction medium. White arrows illustratecell cluster morphology. Black arrows illustrate proteinaceous materialon the surface of clusters cultured in induction medium. Scale bar=5 μm.

FIG. 9A-9D graphically illustrate expression of various feline genes infeline male (open bars) and female (dark bars) ASCs following threestages of pancreatic β cell culture induction. FIG. 9A graphicallyillustrates expression of feline NK6 homeobox 1 (NK6.1) in feline ASCsfollowing three stages of pancreatic β cell culture induction. FIG. 9Bgraphically illustrates expression of feline paired box 6 (Pax6) infeline ASCs following three stages of pancreatic β cell cultureinduction. FIG. 9C graphically illustrates expression of feline ISL LIMhomeobox 1 (Isl1) in feline ASCs following three stages of pancreatic βcell culture induction. FIG. 9D graphically illustrates expression offeline glucose transporter 2 (Glut2) in feline ASCs following threestages of pancreatic β cell culture induction. Expression levels (LSmean±SEM) are shown. Columns with distinct superscripts aresignificantly different between sexes within stages. Different asterisknumbers (*) are significantly different among stages within gender.

FIGS. 10A-10C graphically illustrate expression of various genes infeline male (open bars) and female (dark bars) ASCs following threestages of pancreatic β cell culture induction. FIG. 10A graphicallyillustrates expression of feline insulin in feline ASCs following threestages of pancreatic β cell culture induction. FIG. 10B graphicallyillustrates expression of feline glucagon in feline ASCs following threestages of pancreatic β cell culture induction. FIG. 10C graphicallyillustrates expression of feline somatostatin in feline ASCs followingthree stages of pancreatic β cell culture induction. Expression levels(LS mean±SEM) are shown. Columns with different asterisk numbers (*) aresignificantly different among stages within gender.

FIGS. 11A-11D graphically illustrate expression of various genes infeline male (open bars) and female (dark bars) ASCs following threestages of pancreatic β cell culture induction. FIG. 11A graphicallyillustrates expression of feline proto-oncogene tyrosine-protein kinaseROS1 in feline ASCs following three stages of pancreatic β cell cultureinduction. FIG. 11B graphically illustrates expression of felineserine/threonine kinase 1 AKT1 in feline ASCs following three stages ofpancreatic β cell culture induction. FIG. 11C graphically illustratesexpression of feline Ras-related protein RAB2A in feline ASCs followingthree stages of pancreatic β cell culture induction. FIG. 11Dgraphically illustrates expression of feline hexokinase 1 HK1 in felineASCs following three stages of pancreatic β cell culture induction.Expression levels (LS mean±SEM) are shown.

FIG. 12 shows an image of a western blot confirming insulin productionby feline pancreatic cell clusters generated from adipose derived stemcells by the methods described herein. The blot shows the protein ladderleft, two samples from cell clusters and two samples from paired samplescultured in stromal (basal) medium (left to right).

DETAILED DESCRIPTION

Diabetes mellitus is among the most common feline endocrinopathies,there is no known cure, and the prevalence is steadily increasing.Unregulated glucose levels contribute to serious, permanent healthproblems in cats of all breeds and ages. Exogenous insulinadministration is necessary to maintain healthy glucose levels whenpancreatic β cells cease to function. Therapy is often a complex, timeconsuming process that relies on non-feline insulin formulations.Treatment with xenogeneic insulin is fraught with complications and istime consuming. As many as 30% of cats succumb to consequences of thedisease within 1 year of diagnosis.

As described herein, starting feline cells such as adipose-derivedmultipotent stromal (stem) cells (ASCs) can be transdifferentiated intopancreatic islet cells that secrete insulin in response to glucose. Thecells can be used to create implantable tissue grafts to restore β cellfunction that can be applied in a permanent or removable configuration.The methods and compositions can be used to generate exogenous felineinsulin for direct administration to cats.

The pancreatic islet cells produced using the methods and compositionsdescribed herein can restore natural insulin production after minimallyinvasive implantation of custom tissue grafts generated from individualstem cells. Such custom insulin producing grafts (e.g., made from ASCs)that are conducive to minimally invasive implantation may cure diabetesin cats.

As illustrated herein, feline ASCs isolated from subcutaneous adiposetissue or from reproductive organs have endodermal transdifferentiationcapability. However, feline ASCs from male donors often have distinctmorphology compared to female donors. Induced cells appear to formfunctional clusters based on zinc accumulation secretion of insulin inresponse to glucose stimulation, the presence of intracellular insulin,and the pancreatic β-cell specific gene expression. The methods andcompositions described herein significantly advance the potential toproduce custom feline insulin for exogenous administrations as well as acell-based therapy for temporary or long term restoration of felinepancreatic (3 cell function.

The compositions and methods have been customized for feline cells andhave been designed to replicate in vivo pancreatic formation anddevelopment in cats. In view of species differences, culture methods aremost effective when designed for the target species (Buang et al., Arch.Med. Res. 43: 83-88 (2012); Dubey et al., Repro. Fertil. Devel. 26: 213(2014); Dang et al., Biomed. Res. Ther. 2: 184-192 (2015); Chandra etal., Stem Cells 27: 1941-1953 (2009)).

In the developed a three-stage induction system, N-2 and B-27 canenhance proliferation and protect against reactive oxygen species in theabsence of FBS. FBS can provide growth factors, nutrients and hormonesfor cell proliferation and adhesion. Additionally, there are severalextrinsic factors used in the system that have beneficial effects ondifferentiation of MSCs into insulin producing cells.

Stage I

Stage 1 can involve culturing adult adipose-derived multipotent stromal(stem) cells (ASCs) for about 1-4 days in a first culture mediumcomprising activin A, sodium butyrate, and 2-mercapethanol.

At Stage 1, activin A and sodium butyrate can direct starting cells suchas ASCs to undergo endoderm differentiation.Insulin-transferrin-selenium (ITS), 2-mercaptoethanol, and thesupplements can protect starting cells such as ASCs and can reduce therisk of apoptosis. Ultra-low attachment culture plates or culturevessels can be used to enhance formation of three-dimensional cellaggregates. For example, protein coated culture plates or culturevessels can be used. Examples of proteins that can coat the cultureplates or culture vessels include collagen, fibronectin, laminin,polylysine, poly-ornithine, or a combination thereof.

Activin A is available commercially from various suppliers, for example,from Invitrogen, PeproTech, StemRD, R&D Systems, and other vendors.

In some cases, agents that have activity like activin A can be usedinstead of activin A. Activin A is a member of the TGFβ family firstidentified in late 1980s as an inducer of follicle-stimulating hormone.Activin A is highly conserved in evolution and throughout the animalkingdom. It regulates a variety of biologic processes including cellproliferation, hematopoiesis, wound healing, and fibrosis. Activin Asignals through the activin type I (Alk2, 4, or 7) and type II (ActRIIor ActRIIB) receptors and shares with TGFβ the activation of the Smadcascade. See, Phillips et al., Cytokine Growth Factor Rev. 20(2): 153-64(2009); Werner, Cytokine Growth Factor Rev. 17(3): 157-71 (2006).Examples of TGF-β family members that can be used instead of activin Ainclude the decapentaplegic-Vg-related (DVR) related subfamily ofproteins (including bone morphogenetic proteins and the growthdifferentiation factors), and the TGF-β subfamily.

Activin A and/or other TGFβ family members can be used at a variety ofconcentrations, for example, at about 0.1 nanomolar to about 20nanomolar, or from about 0.5 nanomolar to about 15 nanomolar, or fromabout 1 nanomolar to about 10 nanomolar, or from about 2 nanomolar toabout 8 nanomolar, or about 4 nanomolar.

Agents with activities like sodium butyrate can also be used instead ofsodium butyrate (or in combination with sodium butyrate). Sodiumbutyrate is an inhibitor histone deacetylases (HDACs), which are a classof enzymes that remove acetyl groups from an ε-N-acetyl lysine aminoacid on a histone. Examples of HDAC inhibitors that can be used include,for example, butyrate, small molecular weight carboxylates (e.g., lessthan about 250 amu), hydroxamic acids, benzamides, epoxyketones, cyclicpeptides, and hybrid molecules. (See, for example, Drummond et al., AnnuRev Pharmacol Toxicol 45: 495-528 (2005), (including specific examplestherein) which is hereby incorporated by reference in its entirety).Other examples of negative regulators of type I/II HDACs include: sodiumbutyrate, phenyl butyrate, or butyrate, suberoylanilide hydroxamic Acid(SAHA; also called Vorinostat and MK0683), valproic acid (and othershort chain fatty acids), suramin (e.g., suramin sodium), andcombinations thereof.

The HDAC inhibitor can be employed in the compositions and methodsdescribed herein in a variety of amounts and/or concentrations. Forexample, the HDAC inhibitor can be employed at a concentration of about1 micromolar to about 20 millimolar, or about 10 micromolar to about 15millimolar, or about 50 micromolar to about 10 millimolar, or about 100micromolar to about 10 millimolar, or about 0.5 millimolar to about 2millimolar, or about 1 millimolar in a culture medium solution.

The stage 1 culture medium can include beta-mercaptoethanol at a varietyof concentrations. For example, beta-mercaptoethanol can be employed ata concentration of from about 1 micromolar to about 200 micromolar, orfrom about 5 micromolar to about 100 micromolar, or from about 10micromolar to about 80 micromolar, or at about 50 micromolar in aculture medium solution.

The stage 1 compositions and methods can include use of other agentsincluding insulin-transferrin-selenium (ITS), supplements, proteinsand/or growth factors.

Insulin-transferrin-selenium can be present in various amounts.Insulin-Transferrin-Selenium is available from various commercialvenders. Insulin is typically present in culture media at aconcentration of about 1 μg/ml to about 100 μg/ml, or from about 3 μg/mlto about 50 μg/ml, or from about 5 μg/ml to about 20 μg/ml, or at about10 μg/ml. Transferrin is typically present in culture media at aconcentration of about 5 μg/ml to about 150 μg/ml, or from about 20μg/ml to about 100 μg/ml, or from about 30 μg/ml to about 750 μg/ml, orat about 55 μg/ml. Selenium is typically present in culture media at aconcentration of about 1 μg/ml to about 100 μg/ml, or from about 3 μg/mlto about 50 μg/ml, or from about 5 μg/ml to about 20 μg/ml, or at about10 μg/ml. Selenium can be used at a variety of concentrations, forexample, at about 5 ng/ml to about 200 ng/ml, or from about 20 ng/ml toabout 150 ng/ml, or from about 40 ng/ml to about 100 ng/ml, or fromabout 50 ng/ml to about 80 ng/ml, or at about 67 ng/ml. In some cases, acommercial preparation of insulin-transferrin-selenium can be employed,for example, from Gibco BRL (Gaithersburg, Md.), which is often suppliedin concentrated form. For example, 100-fold concentratedinsulin-transferrin-selenium preparations can be diluted 1:100 intoculture medium.

Examples of proteins that can be included in the stage 1 compositionsand methods include serum albumin, collagen, fibronectin, laminin,polylysine, poly-ornithine, or a combination thereof. Examples of growthfactors that can be included in the stage 1 compositions and methodsinclude hepatocyte growth factor (HGF) and/or fibroblast growth factor(bFGF).

In some cases, the stage 1 compositions and methods can includesupplements such as N-2 supplement, B-27 supplement, or a combinationthereof.

Proteins can be included in the stage 1 compositions at a variety ofconcentrations. For example, proteins (e.g., laminin, collagen, or acombination thereof) can be employed at a concentration of from about0.1 μg/ml to about 100 μg/ml, or from about 1 μg/ml to about 10 μg/ml,or from about 2 μg/ml to about 7 μg/ml, or at about 5 μg/ml in a culturemedium solution. Serum albumin (e.g., BSA) can be employed at aconcentration of from about 0.05% to about 10%, or from about 0.1% toabout 5%, or from about 0.5% to about 2%, or at about 1% in a culturemedium solution.

Growth factors can be included in the stage 1 compositions are a varietyof concentrations. For example, growth factors (e.g., HGF, bFGF, or acombination thereof) can be employed at a concentration of from about0.1 ng/ml to about 200 ng/ml, or from about 1 ng/ml to about 150 ng/ml,or from about 10 ng/ml to about 70 ng/ml, or at about 20 ng/ml to about50 ng/ml in a culture medium solution.

For example, the stage 1 media can include 4 nM avidin A, 1 mM sodiumbutyrate, 50 μM 2-mercapethanol, 1% N-2 supplement, 1% B-27 supplement,5 μg/ml laminin (Corning), 50 ng/ml recombinant human hepatocyte growthfactor (HGF), and 20 ng/ml basic fibroblast growth factor (bFGF).

Stage 2

Stage 2 can induce the endoderm cell clusters formed at Stage 1 toprovide cells of the pancreatic endodermal lineage. Cells at this stagecan be cultured on protein coated culture plates or in protein coatedculture vessels.

Stage 2 can involve culturing the first population of cells for 4 to 8days in a second culture medium comprising insulin-transferrin-selenium,and taurine.

The insulin-transferrin-selenium can be present in various amounts.Insulin-Transferrin-Selenium is available from various commercialvenders. Insulin is typically present in culture media at aconcentration of about 1 μg/ml to about 100 μg/ml, or from about 3 μg/mlto about 50 μg/ml, or from about 5 μg/ml to about 20 μg/ml, or at about10 μg/ml. Transferrin is typically present in culture media at aconcentration of about 5 μg/ml to about 150 μg/ml, or from about 20μg/ml to about 100 μg/ml, or from about 30 μg/ml to about 750 μg/ml, orat about 55 μg/ml. Selenium is typically present in culture media at aconcentration of about 1 μg/ml to about 100 μg/ml, or from about 3 μg/mlto about 50 μg/ml, or from about 5 μg/ml to about 20 μg/ml, or at about10 μg/ml. Selenium can be used at a variety of concentrations, forexample, at about 5 ng/ml to about 200 ng/ml, or from about 20 ng/ml toabout 150 ng/ml, or from about 40 ng/ml to about 100 ng/ml, or fromabout 50 ng/ml to about 80 ng/ml, or at about 67 ng/ml. In some cases, acommercial preparation of insulin-transferrin-selenium can be employed,for example, from Gibco BRL (Gaithersburg, Md.). In some cases, acommercial preparation of insulin-transferrin-selenium can be employed,for example, from Gibco BRL (Gaithersburg, Md.), which is often suppliedin concentrated form. For example, 100-fold concentratedinsulin-transferrin-selenium preparations can be diluted 1:100 intoculture medium.

Taurine, or 2-aminoethanesulfonic acid, is an organic compound that iswidely distributed in animal tissues. It is a major constituent of bileand can be found in the large intestine.

Taurine introduced into the induction medium can facilitate developmentof functional β cells. During the induction process, the induced cellclusters can have a morphology similar to the induced β-cell clustermorphology from other species (see, e.g., Okura et al., J. ArtificialOrgans 12:123-130 (2009)) and may lose the capacity to attach to theplastic culture. Loss of the ability to attach to plastic is onecriterion for identifying mesenchymal stromal cells (MSCs) (Dominici etal., Cytotherapy 8 (2006)). Cells cultured in stromal medium retainedtheir plastic affinity after culture on normal culture plates whilethose in induction medium did not, further confirming differentiationand maturation.

Taurine can be employed in the compositions and methods described hereinin a variety of amounts and/or concentrations. For example, taurine canbe employed at a concentration of about 0.01 mM to about 10 mM, or fromabout 0.03 mM to about 5 mM, or from about 0.05 mM to about 1 mM, orfrom about 0.1 mM to about 0.7 mM, or from about 0.2 mM to about 0.5 mM,or at about 0.3 mM.

The compositions and methods can include use of other agents includingone or more types of sugar, one or more types of protein, one or moretypes of growth factors, one or more types of supplements, andcombinations thereof.

Sugars such as glucose or sucrose can be included in the stage 2compositions and methods. Sugars can be employed in the compositions andmethods described herein in a variety of amounts and/or concentrations.For example, sugars (e.g., glucose) can be employed at a concentrationof about 0.1 mM to about 100 mM, or from about 1 mM to about 50 mM, orfrom about 5 mM to about 30 mM, or from about 10 mM to about 25 mM, orfrom about 15 mM to about 20 mM, or at about 17.5 mM.

Proteins can be included in the stage 1 compositions at a variety ofconcentrations. For example, proteins (e.g., laminin, collagen, or acombination thereof) can be employed at a concentration of from about0.1 μg/ml to about 100 μg/ml, or from about 1 μg/ml to about 10 μg/ml,or from about 2 μg/ml to about 7 μg/ml, or at about 5 μg/ml in a culturemedium solution. Serum albumin (e.g., BSA) can be employed at aconcentration of from about 0.05% to about 10%, or from about 0.1% toabout 5%, or from about 0.5% to about 2%, or at about 1% in a culturemedium solution.

Growth factors can be included in the stage 1 compositions are a varietyof concentrations. For example, growth factors (e.g., HGF, bFGF, or acombination thereof) can be employed at a concentration of from about0.1 ng/ml to about 200 ng/ml, or from about 1 ng/ml to about 150 ng/ml,or from about 10 ng/ml to about 70 ng/ml, or at about 20 ng/ml to about50 ng/ml in a culture medium solution.

In some cases, the stage 1 compositions and methods can includesupplements such as N-2 supplement, B-27 supplement, or a combinationthereof.

For example, the stage 2 media can include serum free medium (SFM 2) (6days): DMEM, 17.5 mM glucose, 1% BSA, 1×ITS, 0.3 mM taurine (ACROSOrganics, Morris Plains, N.J.), 5 μg/mllaminin, 20 ng/ml bFGF, 1% N-2supplement, 1% B-27 supplement, 50 ng/ml HGF;

Stage 3

Stage 3 can involve culturing the second population of cells for 2 to 6days in a third culture medium comprising insulin-transferrin-selenium,taurine, glucagon-like peptide 1 (GLP-1), nicotinamide, pentagastrin,and betacellulin.

The insulin-transferrin-selenium can be used at somewhat higherconcentrations than employed for stages 1 and 2. For example, insulin istypically present in culture media at a concentration of about 1.5 μg/mlto about 150 μg/ml, or from about 3 μg/ml to about 50 μg/ml, or fromabout 7 μg/ml to about 30 μg/ml, or at about 15 μg/ml. Transferrin istypically present in culture media at a concentration of about 7.5 μg/mlto about 200 μg/ml, or from about 25 μg/ml to about 150 μg/ml, or fromabout 50 μg/ml to about 100 μg/ml, or at about 75-80 μg/ml. Selenium canbe used at a variety of concentrations, for example, at about 10 ng/mlto about 400 ng/ml, or from about 20 ng/ml to about 300 ng/ml, or fromabout 40 ng/ml to about 200 ng/ml, or from about 50 ng/ml to about 150ng/ml, or at about 100 ng/ml. In some cases, a commercial preparation ofinsulin-transferrin-selenium can be employed, for example, from GibcoBRL (Gaithersburg, Md.). In some cases, a commercial preparation ofinsulin-transferrin-selenium can be employed, for example, from GibcoBRL (Gaithersburg, Md.), which is often supplied in concentrated form.For example, 100-fold concentrated insulin-transferrin-seleniumpreparations can be diluted 1.5:100 into culture medium.

Taurine can be used in amounts that are somewhat greater than theamounts used for Stage 2. For example, taurine can be used in amountsthat are about two-fold, or five-fold, or ten-fold, or bout fifteen-foldgreater than the amounts used for Stage 2. Taurine can be employed inthe stage 3 compositions and methods at a concentration of about 0.005mM to about 20 mM, or from about 0.01 mM to about 15 mM, or from about0.05 mM to about 10 mM, or from about 0.1 mM to about 5 mM, or fromabout 0.2 mM to about 1 mM, or at about 3 mM.

The glucagon-like peptide 1 (GLP-1) can be employed in the compositionsand methods described herein in a variety of amounts and/orconcentrations. For example, the GLP-1 can be employed at aconcentration of about 1 nanomolar to about 300 nanomolar, or about 10nanomolar to about 200 nanomolar, or about 30 nanomolar to about 150nanomolar, or about 50 nanomolar to about 120 nanomolar, or at about 100nanomolar in a culture medium solution.

Nicotinamide is a polyADP-ribose synthetase inhibitor. Other types ofpolyADP-ribose synthetase inhibitors can be used instead of or incombination with nicotinamide. Examples of polyADP-ribose synthetaseinhibitors therefore include nicotinamide, 3-aminobenzamide,1,5-isoquinolinediol and combinations thereof. The polyADP-ribosesynthetase inhibitors (e.g., nicotinamide) can be employed at aconcentration of about 0.05 millimolar to about 100 millimolar, or about0.1 millimolar to about 20 millimolar, or about 0.3 millimolar to about10 millimolar, or about 0.5 millimolar to about 5 millimolar, or about 1millimolar in a culture medium solution.

Pentagastrin is a synthetic polypeptide that has effects like gastrin.When administered, it can stimulate the secretion of gastric acid,pepsin, and intrinsic factor. Pentagastrin can be employed in thecompositions and methods described herein in a variety of amounts and/orconcentrations. For example, pentagastrin can be employed at aconcentration of from about 0.1 nanomolar to about 30 nanomolar, orabout 1 nanomolar to about 20 nanomolar, or about 3 nanomolar to about15 nanomolar, or about 5 nanomolar to about 12 nanomolar, or at about 10nanomolar in a culture medium solution.

Betacellulin is a member of the EGF family of growth factors. It issynthesized primarily as a transmembrane precursor, which is thenprocessed to mature molecule by proteolytic events. It can be a ligandfor EGF receptor Betacellulin can be employed in the compositions andmethods described herein in a variety of amounts and/or concentrations.For example, betacellulin can be employed at a concentration of fromabout 0.1 ng/ml to about 200 ng/ml, or from about 1 ng/ml to about 100ng/ml, or from about 3 ng/ml to about 50 ng/ml, or from about 5 ng/ml toabout 25 ng/ml, or at about 100 ng/ml. in a culture medium solution.

The stage 3 compositions and methods can include use of other agentssuch as sugars, proteins, supplements, and amino acids.

Sugars such as glucose or sucrose can be included in the stage 2compositions and methods. Sugars can be employed in the compositions andmethods described herein in a variety of amounts and/or concentrations.For example, sugars (e.g., glucose) can be employed at a concentrationof about 0.1 mM to about 100 mM, or from about 1 mM to about 50 mM, orfrom about 5 mM to about 30 mM, or from about 10 mM to about 25 mM, orfrom about 15 mM to about 20 mM, or at about 17.5 mM.

Proteins can be included in the stage 1 compositions at a variety ofconcentrations. For example, proteins (e.g., laminin, collagen, or acombination thereof) can be employed at a concentration of from about0.1 μg/ml to about 100 μg/ml, or from about 1 μg/ml to about 10 μg/ml,or from about 2 μg/ml to about 7 μg/ml, or at about 5 μg/ml in a culturemedium solution. Serum albumin (e.g., BSA) can be employed at aconcentration of from about 0.05% to about 10%, or from about 0.1% toabout 5%, or from about 1% to about 3%, or at about 1.5% in a culturemedium solution.

Growth factors can be included in the stage 1 compositions are a varietyof concentrations. For example, growth factors (e.g., HGF, bFGF, or acombination thereof) can be employed at a concentration of from about0.1 ng/ml to about 200 ng/ml, or from about 1 ng/ml to about 150 ng/ml,or from about 10 ng/ml to about 70 ng/ml, or at about 20 ng/ml to about50 ng/ml in a culture medium solution.

In some cases, the stage 1 compositions and methods can includesupplements such as N-2 supplement, B-27 supplement, or a combinationthereof.

In some cases, the third stage compositions and methods can include useof the following serum free media (SFM 3) for 4 days: DMEM, 17.5 mMglucose, 1.5% BSA, 1.5×ITS, 3 mM taurine, 100 nM glucagon-like peptide 1(GLP-1), 1 mM nicotinamide, 1×non-essential amino acids (NEAA, e.g.,from Gibco), 10 nM pentagastrin, 1% N-2 supplement, 1% B-27 supplement,50 ng/ml HGF, 20 ng/ml bFGF, 5 μg/ml laminin, 10 ng/ml betacellulin.

At a third stage, the cell clusters are converted into functionalpancreatic hormone-expressing islet-like cell aggregates with the helpof glucagon-like peptide 1 (GLP-1), nicotinamide, taurine, andβ-cellulin. Nicotinamide can promote the maturation of precursor cellsinto insulin-producing cells and increase the rate of proinsulinbiosynthesis. Addition of β-cellulin and nicotinamide to the Stage 3induction medium can promote (3 cell maturation and can generate cellsthat express endocrine hormones including insulin and glucagon.Theophylline can also be used in the Stage 3 induction medium.

As illustrated herein, the induced cell clusters produced during stage 3were specifically labeled with the zinc-chelating dye dithizone (DTZ),which stains β-cells due to the presence of zinc in insulin-containingsecretory granules (D'Amour et al., Nature Biotech. 24: 1392 (2006)).Immunofluorescence, DTZ staining, and immune electron microscopy resultsconfirmed that the induced cells produced using the compositions andmethods described herein expressed insulin. As shown herein, RT-PCRshowed that the induced cells coexpressed insulin, glucagon, andsomatostatin. These results are in accordance with normal pancreaticdevelopment, in which immature islets are known to coexpress pancreatichormones (Chandra et al. Stem Cells 27: 1941-1953 (2009)).

Male/Female

Surprisingly, the mRNA levels of Pax6 and Glut2 were different in maleand female ASCs at such an early induction stage, indicating that thecells from male cats may have higher sensitivity than that from femalecats. For example, human male ASCs may have greater osteogenic potentialthan female ASCs. Pax6 is a transcription factor that can transactivatethe insulin promoters (Sander et al., Genes & development 11:1662-1673(1997)). The higher expression of Pax6 in male ASCs at early inductionstages may indicate that the male ASCs undergo pancreatic β celldifferentiation more easily than female.

The results described herein show that male and female ASCs haveendodermal transdifferentiation capability. The induced cell clusterscan secrete insulin with the glucose stimulation. In the clinicalcontext, these results demonstrate the potential for stem cell-basedtherapy to treat feline diabetes. The mechanisms of thetransdifferentiation in feline ASCs are not clear. Hence, it may not bepossible to predict what compositions and methods would successfullytransdifferentiate feline starting cells into cells that can secreteinsulin cells. However, the methods and compositions described herein dotransdifferentiate feline ASCs into functional insulin-producing cells,demonstrating that such methods and compositions are provide a viabletherapeutic option for feline diabetes.

Kits

Also provided are kits for generating pancreatic precursor cells and/orpancreatic cells. The kits can contain any of the compositions describedherein and instructions for using the compositions for generatingdefinitive endoderm cells and/or pancreatic precursor cells and/orpancreatic cells. Each of the compositions can be separately packaged.Each composition can contain any of the compounds or proteins describedherein at a concentration that is convenient for addition to a cultureof cells. For example, compositions can be concentrated to about 10×,50×, 100×, or 1000× of the concentration at which it would be employedto pancreatic precursor cells and/or cells that can secrete insulincells and/or pancreatic beta cells. The instructions can provideguidance for appropriate addition (dilution) of the compositions into acell culture, and/or guidance on other culture conditions (e.g.,appropriate cell culture media, an appropriate duration of exposure tothe compositions, etc.). The instructions can also provide guidance onthe selection of starting cells for generating pancreatic precursorcells and/or cells that can secrete insulin cells and/or pancreaticcells. In addition, the instructions can provide information for testingand/or recognition of the generated pancreatic precursor cells and/orcells that can secrete insulin cells and/or pancreatic cells.

The kits can also provide components and instructions for administeringpancreatic precursor cells and/or cells that can secrete insulin cellsand/or pancreatic beta cells to mammalian (e.g., feline) subjects. Theinstructions can provide guidance on the numbers and the type(s)(phenotype) of cells to be administered. The instructions can alsoprovide instructions for administration of pancreatic precursor cellsand/or cells that can secrete insulin cells and/or pancreatic beta cellsby surgical implantation or by infusion. For example, the kits canprovide diluents, pharmaceutically acceptable carriers, scalpels,syringes, catheters, bandages, antiseptics, and the like to permitadministration of cells.

Mixtures

The pancreatic precursor cells and/or cells that can secrete insulincells and/or pancreatic cells can be present in any of the foregoingcompositions. The pancreatic precursor cells and/or cells that cansecrete insulin cells and/or pancreatic cells can also be present in atherapeutically acceptable carrier such as saline, phosphate bufferedsaline, or other aqueous carrier. Such a combination of the compositionsdescribed herein, or a therapeutically acceptable carrier, withpancreatic precursor cells and/or cells that can secrete insulin cellsand/or pancreatic cells can be referred to as a mixture.

The mixtures can contain about 1 to about 10¹⁰ pancreatic precursorcells and/or cells that can secrete insulin cells and/or pancreatic(e.g., beta) cells.

The pancreatic precursor cells and/or cells that can secrete insulincells and/or pancreatic cells generated as described herein can beisolated, separated, or purified from culture media, compositions, orother mixtures in which they are generated. The pancreatic precursorcells and/or cells that can secrete insulin cells and/or pancreaticcells generated as described herein can be enriched or cultured toincrease the proportion or numbers of desired cells in the population.Any such isolate, separation, purification, enrichment, or culture is amixture as described herein.

An isolating step can include providing the cells in the cell culturewith a reagent which binds to a marker expressed in the desired celltype (e.g., pancreatic precursor cells, and/or cells that can secreteinsulin cells and/or pancreatic cells) but which is not substantiallyexpressed in other cells present in the cell culture. The reagent-boundcells can be separated from the non-reagent-bound cells by numerousmethods. For example, an antibody against a marker that is selectivelypresent on the desired cells can be provided to cells in a cell culture.Antibody-bound cells can then be separated from other cells in theculture by, for example, fluorescent activated cell sorting (FACS),binding the antibody to a solid support or isolating appropriatelytagged antibody in a magnetic field. In some embodiments, the antibodyis released from the cells after the separation process.

As an alternative means of separation, at least some of the desiredcells can be separated from at least some of the other cells in theculture by specifically fluorescently labeling the desired cells inculture and then separating the labeled cells from the unlabeled cellsby FACS.

An enriched cell population of pancreatic progenitor cells, and/or cellsthat can secrete insulin cells, and/or pancreatic beta cells produced,for example, by an isolating step can be substantially free of cellsother than pancreatic progenitor cells, and/or cells that can secreteinsulin cells and/or pancreatic beta cells. In other embodiments, theenriched cell populations can have at least about 50% to at least about100% pancreatic progenitor cells, and/or cells that can secrete insulincells and/or pancreatic beta cells. In still other embodiments, theenriched cell populations comprise from at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 99%, or at leastabout 100% pancreatic progenitor cells, and/or cells that can secreteinsulin cells, and/or pancreatic beta cells.

In some instances, the pancreatic progenitor cells, cells that cansecrete insulin cells, and/or pancreatic beta cells are expanded, forexample, by culturing the cells under conditions that permit celldivision. For example, some embodiments include a culturing step thatcomprises plating a cell population on a surface such as a cultureplate. In some embodiments, the cells are plated on a surface coatedwith a protein, poly-amino acid or carbohydrate (e.g., collagen,fibronectin, laminin, polylysine, poly-ornithine, or a combinationthereof).

In other embodiments, the culturing step comprises incubating the cellpopulation or portion thereof in an expansion medium comprising about 2%(v/v) serum. In some embodiments, the serum concentration can range fromabout 0% (v/v) to about 20% (v/v). For example, in some methodsdescribed herein, the serum concentration of the medium can be about0.05% (v/v), about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about0.8% (v/v), about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3%(v/v), about 4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v),about 8% (v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) orabout 20% (v/v). In some embodiments, serum replacement is included inthe medium, and no serum is employed.

Using the methods described herein, cell populations or cell culturescan be enriched in pancreatic progenitor (precursor) cells, cells thatsecrete insulin, and/or pancreatic beta cell content by at least about2-fold to about 1000-fold as compared to cell populations or cellcultures produced by the methods and compositions described herein. Insome embodiments, pancreatic progenitor (precursor) cells, cells thatsecrete insulin, and/or pancreatic beta cells can be enriched by atleast about 5-fold to about 500-fold as compared to cell populations orcell cultures produced by the methods and compositions described herein.In other embodiments, pancreatic progenitor, cells that secrete insulin,and/or pancreatic beta cells can be enriched from at least about 10- toabout 200-fold as compared to cell populations or cell cultures producedby the methods and compositions described herein. In still otherembodiments, pancreatic progenitor, cells that secrete insulin, and/orpancreatic beta cells can be enriched from at least about 20- to about100-fold as compared to cell populations or cell cultures produced bythe methods and compositions described herein. In yet other embodiments,pancreatic progenitor, cells that secrete insulin, and/or pancreaticbeta cells can be enriched from at least about 40- to about 80-fold ascompared to cell populations or cell cultures produced by the methodsand compositions described herein. In certain embodiments, pancreaticprogenitor, cells that secrete insulin, and/or pancreatic beta cells canbe enriched from at least about 2-to about 20-fold as compared to cellpopulations or cell cultures produced by the methods and compositionsdescribed herein.

Some embodiments described herein relate to cell cultures or cellpopulations comprising from at least about 5% pancreatic progenitor,cells that secrete insulin, and/or pancreatic beta cells to at leastabout 95% pancreatic progenitor, cells that secrete insulin, and/orpancreatic beta cells. In some embodiments the cell cultures or cellpopulations comprise mammalian cells. In preferred embodiments, the cellcultures or cell populations comprise feline cells. For example, certainspecific embodiments relate to cell cultures comprising feline cells,wherein from at least about 5% to at least about 95% of the cells arepancreatic progenitor, cells that secrete insulin, and/or pancreaticbeta cells. Other embodiments relate to cell cultures comprising felinecells, wherein at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90% or greater than 90% of the cells are pancreaticprogenitor, cells that secrete insulin, and/or pancreatic beta cells. Inembodiments where the cell cultures or cell populations comprisemammalian feeder cells, the above percentages are calculated withoutrespect to the feeder cells in the cell cultures or cell populations.

Therapy

Also described herein is a method for treating a subject (e.g., a felinesubject) suffering from, or at risk of developing, diabetes. This methodinvolves generating pancreatic progenitor, cells that secrete insulin,and/or pancreatic beta cells as described herein, and administering orimplanting the cells into a mammalian subject (e.g., a feline subject).

The pancreatic progenitor, cells that secrete insulin, and/or pancreaticbeta cells can be implanted as dispersed cells or formed into clusters.Alternatively, pancreatic progenitor, cells that secrete insulin, and/orpancreatic beta cells can be infused into the subject, for example, viaa hepatic portal vein. Alternatively, cells may be provided inbiocompatible degradable polymeric supports, porous non-degradabledevices or encapsulated to protect from host immune response. Cells maybe implanted into an appropriate site in a subject. The implantationsites include, for example, the liver, natural pancreas, renalsubcapsular space, omentum, peritoneum, subserosal space, intestine,stomach, or a subcutaneous pocket.

The amount of cells used in implantation depends on a number of variousfactors including the subject's condition and response to the therapy,and can be determined by one skilled in the art. For example, the numberof cells administered can range from about 1000 to about 10⁹, or fromabout 1000 to about 10⁸, or from about 1000 to about 10⁷, or from about1000 to about 10⁶, or from about 10000 to about 10⁷.

In one aspect, a method is provided for treating a subject sufferingfrom, or at risk of developing diabetes. This method involves culturinga starting cell population, differentiating or redirecting the culturedcells in vitro into a first population of cells that express at leastone of the following genes: Nkx 6.1, Pax6, Isl1, or Glut-2;differentiating the first population of cells into a second populationcontaining pancreatic progenitor cells and administering the secondpopulation of cells to a subject. In some instances pancreaticprogenitor cells are enriched within the second population or purifiedfrom the second population of cells to generate a third population ofcells that is substantially free of non-pancreatic cells.

The cells to be administered can be incorporated into athree-dimensional support. The cells can be maintained in vitro on thissupport prior to implantation into the subject. Alternatively, thesupport containing the cells can be directly implanted in the subjectwithout additional in vitro culturing. The support can optionally beincorporated with at least one pharmaceutical agent that facilitates thesurvival and function of the transplanted cells.

Support materials suitable for use include tissue templates, conduits,barriers, and reservoirs useful for tissue repair. In particular,synthetic and natural materials in the form of foams, sponges, gels,hydrogels, textiles, and nonwoven structures, which have been used invitro and in vivo to reconstruct or regenerate biological tissue, aswell as to deliver chemotactic agents for inducing tissue growth, aresuitable for use in practicing the methods described herein. See, forexample, the materials disclosed in U.S. Pat. Nos. 5,770,417, 6,022,743,5,567,612, 5,759,830, 6,626,950, 6,534,084, 6,306,424, 6,365,149,6,599,323, 6,656,488, U.S. Published Application 2004/0062753 A1, U.S.Pat. Nos. 4,557,264 and 6,333,029, each of which is specificallyincorporated by reference herein in its entirety.

The mammalian subject can be a domestic animal, or a laboratory animal.In some cases, the subject is a feline subject.

Definitions

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a compound,” “a cell,” “anucleic acid” or “a polypeptide” includes a plurality of such compounds,cells, nucleic acids or polypeptides (for example, a solution of cells,nucleic acids or polypeptides, a suspension of cells, or a series ofcompound, cell, nucleic acid or polypeptide preparations), and so forth.

The following Examples describe some experimental work performed duringdevelopment of the invention. Appendix A may provide additionalinformation about the invention.

Example 1: Materials and Methods This Example Describes Some of theMaterials and Methods Employed in the Development of the InventionFeline ASC Isolation

Feline ASCs were isolated during routine sterilization of 8 male and 8female adult cats. Adipose tissue was isolated with sharp dissection,minced, and digested in type I collagenase (33 ml/g adipose tissue,Worthington Biochemical, Lakewood, N.J.) within 4 h of harvest. Tissuesfrom each cat (n=5) were divided into three equal portions, and eachportion was digested by one of three methods: 1) 0.1% type I collagenasein DMEM, 0.5 h, 60 rpm (Classic); 2) 0.3% type I collagenase in Kreb'sRinger buffer (KRB), 0.5 h, 1,000 rpm stirring (New); and 3) 0.3% type Icollagenase in KRB, 1 h, 1,000 rpm stirring (Hour). Tissue was added tocollagenase solution in a 30 ml glass jar and stirred with a stir bar at37° C. for the New and Hour digestion methods. For the Classic digestionmethod, digestion mixtures within glass jars were agitated on athree-dimensional plate shaker at 37° C. Digests were filtered and thencentrifuged (260 g, 5 min). Resulting SVF pellets were resuspended in 5ml red cell lysis buffer (0.16 mol/L NH₄Cl, 0.01 mol/L KHCO₃, 0.01%EDTA) followed by cell seeding in 10 mm cell culture dishes in stromalmedium (DMEM-Ham's F12, 10% fetal bovine serum (FBS, Hyclone), 1%antibiotic/antimycotic solution). Medium was refreshed after overnightincubation under standard conditions (37° C., 5% CO₂) and then every 2-3days. The total number of colonies with 20 or more cells was determinedafter 7 days of culture. MSC density was then calculated as colonynumber/adipose weight (g). The New method was selected to isolate ASCsfor the remainder of the study based on the highest number of colonies/gtissue. When SVF cells reached 80% confluence, they were detached with0.25% trypsin and 0.1% EDTA. Cells were seeded at a density of 5×10³cells/cm² for P0 and all subsequent passages for evaluation of freshcells. To prepare revitalized cells, aliquots (5×10⁵ cells) of P0 cellswere frozen in cryopreservation medium (80% FBS, 10% DMEM, 10% dimethylsulfoxide) in liquid nitrogen for 30 days. Cells were then revitalizedand seeded at a density of 5×10³ cells/cm² in stromal medium.Revitalized cells were subsequently cultured and evaluated identicallyto fresh cells.

Study Design

For the purposes of this study, the stromal vascular fraction was theprimary cell isolate, and passage (P) 0 was the first cell passage.Fresh cells were expanded to P3 and then paired samples were cultured instromal or three-step islet cell induction medium. The zincconcentration, insulin production, and ultrastructure was comparedbetween stromal cells and the induced cells from stage 3. Multipotentcapacity and lineage specific gene expression (insulin, NK6 homeobox 1(NK6.1), proto-oncogene tyrosine-protein kinase ROS1 (ROS1),somatostatin (STS), ISL LIM homeobox 1 (Isl1), glucagon (GCG), pairedbox 6 (Pax6), AKT serine/threonine kinase 1 (AKT1), Ras-related proteinRab-2A (RAB2A), hexokinase 1 (HK1), and glucose transporter 2 (Glut2))were compared between culture conditions in male and female donors. Allmaterials and reagents were from Sigma-Aldrich, St. Louis, Mo. unlessotherwise noted.

Cell Isolation

Tissue was minced and digested with 0.3% type I collagenase (WorthingtonBiochemical Corporation, Lakewood, N.J.) in Kreb's Ringer buffer (KRB)for 30 minutes, 1,000 rpm stirring with a stir bar at 37° C. Afterfiltering (100 μm nylon cell strainers, BD Falcon, Bedford, Mass.) andcentrifugation (260×g, 5 min), the resulting SVF pellets wereresuspended in 5 ml red blood cell lysis buffer (0.16 mol/L NH4Cl, 0.01mol/L KHCO3, 0.01% ethylenediaminetetraacetic acid (EDTA)) for 5 min.The SVF was collected after centrifugation (260×g, 5 min) and seeded in10-mm cell culture dishes in stromal medium (Dulbecco's modified Eagle'smedium F-12 (DMEM/F-12, Hyclone, Logan, Utah), 1% antibiotic/antimycoticsolution (MP Biomedical, Irvine, Calif.), 10% fetal bovine serum (FBS,Hyclone)). Stromal medium was refreshed after 24 hours and then every 3days. After 70-80% confluence, the SVF cells were detached with 0.05%trypsin (Hyclone) and cells were seeded in T75 flasks with the densityof 5×103 cells/cm2 for P0 and all subsequent passages. Proceduresperformed at temperatures other than room temperature are indicated.

Multipotentiality

Cell isolates were tested to confirm multipotentiality, P1 fibroblasticcolony formation and adipogenic and osteogenic differentiation. Forfibroblastic colony formation, P1 cells were cultured in stromal mediumfor 7 days, fixed with 4% formalin, and stained with 0.1% toluidineblue. To assess adipogenesis, cells were cultured in stromal medium to70-80% confluence, washed with phosphate buffered saline (PBS, Hyclone)and then cultured in adipogenic medium (Table 1) for 10 days. They werethen fixed with 4% neutral paraformaldehyde (PFA) and stained with oilred O. For osteogenesis, the cells were cultured in stromal medium asabove, then in osteogenic preinduction medium for 10 days followed byosteogenic induction medium (Table 1) for another 10 days. Colonies werefixed with 70% ice cold ethanol and stained with 2% alizarin red.

Differentiation of Feline ASCs into IPCs

Cell isolates were culture expanded to P3. At 70-80% confluence, theywere seeded in 6-well ultra-low attachment plates (Corning, Corning,N.Y.) at 1×10⁶ cells/well and cultured for 7 days in stromal medium. Athree-stage protocol was used to induce β-cell islet-like clusters(Table 1). The cells in ultra-low attachment plates were incubated inserum free medium (SFM) 1 for 2 days as stage 1 and then in SFM 2 foranother 6 days at stage 2. Cells were then manually transferred to astandard 6-well plate (Thermo Fisher Scientific, Waltham, Mass., USA)and cultured in stage 3 induction medium for another 4 days in standard6-well plates. All media were refreshed every 2 days and the medium wasrefreshed every 24 hours in stage 3. Paired cells were cultured instromal medium in standard 6-well plates throughout the inductionprocess.

TABLE 1 Composition of induction medium for differentiation MediumComposition Adipogenic Minimum essential medium alpha (α-MEM), 10%rabbit serum, medium 10% FBS, 10 nM dexamethasone, 5 μg/ml insulin, 50μM 5,8,11,14-eicosatetraynoic acid (ETYA, Cayman, Ann Arbor, MI), 100 μMindomethacin Osteogenic DMEM, 10% FBS, 100 nM dexamethasone, 0.25 mML-ascorbic preinduction acid medium Osteogenic Osteogenic preinductionmedium supplemented with 10 mM β- induction glycerophosphate mediumβ-cell induction Stage 1: medium SFM 1 (2 days): DMEM, 17.5 mM glucose,1% BSA (bovine serum albumin), 1 × insulin-transferrin-selenium (ITS,Gibco BRL, Gaithersburg, MD), 4 nM activin A (R&D Systems Inc.,Minneapolis), 1 mM sodium butyrate, 50 μM 2-mercapethanol, 1% N-2supplement (R&D Systems Inc.), 1% B-27 supplement (Gibco), 5 μg/mllaminin (Corning), 50 ng/ml recombinant human hepatocyte growth factor(HGF, EMD Millipore, Temecula, CA), 20 ng/ml basic fibroblast growthfactor (bFGF, Gibco); Stage 2: SFM 2 (6 days): DMEM, 17.5 mM glucose, 1%BSA, 1 × ITS, 0.3 mM taurine (ACROS Organics, Morris Plains, NJ), 5μg/ml laminin, 20 ng/ml bFGF, 1% N-2 supplement, 1% B-27 supplement, 50ng/ml HGF; Stage 3 SFM 3 (4 days): DMEM, 17.5 mM glucose, 1.5% BSA, 1.5× ITS, 3 mM taurine, 100 nM glucagon-like peptide 1 (GLP-1, TOCRISbioscience, Ellisville, MO), 1 mM nicotinamide (ACROS Organics), 1 ×non-essential amino acids (NEAA, Gibco), 10 nM pentagastrin (TOCRISbioscience), 1% N-2 supplement, 1% B-27 supplement, 50 ng/ml HGF, 20ng/ml bFGF, 5 μg/ml laminin, 10 ng/ml betacellulin (R&D Systems)

Dithizone Staining—Zinc Concentration

Following the induction process, cells were incubated with dithizone(DTZ, Fisher Scientific, Fairlawn, N.J.) solution (10 μl DTZ workingsolution (1 mg/ml in dimethyl sulfoxide (Fisher Scientific)) in 1 ml ofthe culture medium) for 30 min at 37° C. Cells were washed in PBS afterincubation and imaged with light microscope.

Immunohistochemistry—Intra-Cellular Insulin

The Dylight 633 antibody labeling kit (Thermo Fisher Scientific,Somerset, N.J.) was used to label the antibody goat anti insulinaccording to the manufacturer's instruction. Briefly, 100 μl antibodysolution (1 mg/ml) was mixed with 8 μl supplied borate buffer and themixture was incubated with Dylight Reagent for 60 min and protected fromlight. The labeling reaction was mixed with resin and then centrifuged(1000×g, 1 min) to collect the labeled antibody.

Cells were washed with PBS and fixed overnight in 4% neutral PFA. Theywere permeabilized with 0.5% Triton X-100 in PBS and then incubated withthe labeled antibody goat anti insulin (1:200 in PBS, Santa CruzTechnologies, CA) for 30 min. After incubation, the cells were washedwith PBS and then cytoskeletal actins was stained with β actin-FITC for30 min (1:500 in PBS, Neomarkers, Fremont, Calif.). Photomicrographswere obtained for all labeled cells with confocal laser scanningmicroscopy (CLSM, Leica TCS SP2, Leica, Wetzlar, Germany).

Glucose Challenge Assay—Glucose Sensitivity

The induced islet-like cell clusters were collected and washed twicewith PBS. The islet-like cell clusters were incubated with KRB bufferfor 1 hour at 37° C., followed by incubation with KRB buffersupplemented with different glucose concentrations (25 and 55 mM) for 30min at 37° C. Following incubation, the medium was collected and storedat −80° C. until further use. The stored medium was analyzed for insulincontent using a feline specific enzyme-linked immunosorbent assay(ELISA) kit (Mybiosource, San Diego, Calif.). The cells cultured instromal medium were used as a control.

Transmission Electron Microscopy—Ultrastructure

Following a PBS rinse, cells were fixed in 2% PFA and 2.5%glutaraldehyde in 0.1 M PBS for 10 min. The samples were centrifuged(350×g, 5 min) and then fixed with fresh fixative with gentle agitationfor 2 hours. They were mixed with equal amounts of 3% agarose and themixture was placed to a glass slide. When the mixture solidified, it wassliced into cubes. The cubes were washed with 0.1 M PBS and 0.08Mglycine 5 times (15 min/time) followed by incubation with 2% osmiumtetroxide in 0.1 M PBS in the darkness for 1 hour to fix the cells. Thesamples were washed with H₂O and dehydrated in a series ofethanol-distilled water solutions. The dehydrated samples wereinfiltrated with 1:1 ethanol and LR white resin for 2 hours, and theninfiltrated with 100% LR white resin for another 2 hours. Embeddedsamples were placed into the bottom of a beem capsule and incubated inan 18° C. oven for 24 hours. Ultra-thin sections (90 nm) were cut andstained with 2% uranyl acetate and lead citrate. Some sections weredirectly evaluated with transmission electron microscopy (JEOL JEM-1400,Japan). The other sections were blocked in 5% BSA in PBS for 30 min andthen incubated with goat anti insulin in 0.1% BSA in PBS (1:20) foranother 90 min. After incubation, the sections were washed in 0.1% BSAin PBS 6 times (5 min/time) and then incubated in secondary antibody(rabbit anti goat IgG-Gold, Sigma-Aldrich) in 0.1% BSA in PBS foranother 90 min. After incubation, the sections were washed with 0.1% BSAbuffer and PBS, respectively. The sections were then fixed in 2%glutaraldehyde in PBS for 5 min and contrasted with 2% uranyl acetateand lead citrate after being thoroughly washed in distilled water. Goldlabeled sections were observed with TEM.

Scanning Electron Microscopy—Surface Ultrastructure

Cells were collected by filtration and fixed in 2% PFA and 2%glutaraldehyde in 0.1 M PBS for 15 min. The solution was extracted intoa 10 ml syringe with a Swinney filter holder fitted with a 2 μm porepolycarbonate with 13 mm diameter. Entrapped cells on the filtered werefixed as before another 15 min and then rinsed with 0.1 M PBS anddistilled water. The filter was removed from the syringe and dried withhexamethyldisilazane (HMDS, Electron Microscopy Sciences, FortWashington, Pa.) for 30 min, 1:1 100% ethanol and HDMS, and 2 changesfor 30 min each with 100% HDMS. Finally the HDMS was removed and thesamples were placed in a hood overnight to air-dry. The dried sampleswere mounted onto aluminum SEM stubs, coated with platinum in an EMS550X sputter coater and imaged with JSM-6610 High vacuum mode SEM (JEOLLtd., Japan).

RT-PCR—Gene Expression

Total RNA was isolated from cells harvested from each induction stage(EZNA® MicroElute Total RNA kit, Omega, Bio-Tek, Norcross, Ga.). Thequality and concentration was determined spectrophotometrically(NanoDrop ND-1000; NanoDrop Technologies, Wilmington, Del.), and cDNAsynthesized (Maxima First-Strand cDNA synthesis kit, Thermo Scientific,Waltham, Mass.). Feline pancreatic target gene levels (insulin, Isl1,HK1, Glut-2, NK6.1, ROS1, STS, GCG, Pax6, AKT1, and RAB2A) (Table 2)were quantified with real-time RT-PCR using the Thermo Fisher Absolute™Blue QPCR Rox Mix technology and an ABI Prism 7900 HT Sequence DetectionSystem (Applied Biosystems, Foster City, Calif.) using feline-specificprimers. The ΔCt values were determined relative to the reference geneβ-actin.

TABLE 2 Primer Sequences Lineage Primer Sequences Accession No.Housekeeping β-actin F: AGCCTTCCTTCCTGGGTATG XM_006941899.3 SEQ ID NO: 1R: ACAGCACCGTGTTAGCGTAG SEQ ID NO: 2 Transcription Nkx 6.1F: AACGAAATACTTGGCGG XM_019829291.1 Factor SEQ ID NO: 3R: CCAGAGGCTTGTTGTAGTCG SEQ ID NO: 4 Transcription Pax6F: GGCAATCGGTGGTAGTAA XM_019812231.1 Factor SEQ ID NO: 5R: CTTGGTATGTTATCGTTGG SEQ ID NO: 6 Transcription Isl1F: CAAGGACAAGAAGCGGAG XM_003981424.3 Factor SEQ ID NO: 7R: CTGGGTTTGCCTGTAAGC SEQ ID NO: 8 Transcription Glut 2F: TTGGCTTGGATGAGTTACG XM_003991916.3 Factor SEQ ID NO: 9R: GACTTTCCTTTGGTTTCCG SEQ ID NO: 10 Protein InsulinF: CTTCGTCAACCAGCACC XM_019811180.1 SEQ ID NO: 11 R: ACAGCATTGCTCCACGASEQ ID NO: 12 Glucagon F: TGAACACCAAGAGGAACAA XM_006935320.2SEQ ID NO: 13 R: ACCAGCCAAGCAATGAAT SEQ ID NO: 14 Somato-F: CCAGACAGAGAACGATGCC XM_003991805.4 statin SEQ ID NO: 15R: CAGGGTTTGAGTTAGTGGA SEQ ID NO: 16 Oncogene ROS1F: AACAACAGCCTCTACTACAG XM_019831130.1P SEQ ID NO: 17R: TATCCTCCGACCGAATCC SEQ ID NO: 18 Akt1 F: CCAACACCTTCATCATCCGNM_001322435.1 SEQ ID NO: 19 R: CCATCATTTCCTCCTCCTG SEQ ID NO: 20 RAB2AF: ACAGACAAGAGGTTTCAGC XM_019822712.1 SEQ ID NO: 21R: TATGACCGTGTGATGGAAC SEQ ID NO: 22 HK1 F: TGAGAAGATGGTGAGTGGCXM_006937834.3 SEQ ID NO: 23 R: GGCAGAGCGAAATGAGAC SEQ ID NO: 24

Statistical Analysis

All results are presented as least squares (LS) mean±SEM. Statisticalanalyses were performed with the JMP statistical package (v 13.0.0, SASInstitute Cary, N.C.). Mixed ANOVA models were used to evaluate insulinlevels between glucose concentrations within genders and between genderswithin glucose concentrations. The same models were used to evaluatetarget gene expression among induction stages within genders and betweengenders within induction stages. Tukey's post hoc tests were applied formultiple group comparisons (p<0.05).

Example 2: ASC Multipotentiality

All ASC isolates following culture in stromal medium displayedosteogenic and adipogenic differentiation based on histochemicalstaining. Cells had a fibroblastic shape when cultured in stromal medium(FIG. 1A). Colony calcium stained with alizarin red following culture inosteogenic medium (FIG. 1B), and lipid droplets stained with oil red Oafter adipogenic medium culture (FIG. 1C).

Example 3: Cell Morphology

All ACSs cultured in ultralow attachment plates formed cell clusters.Following transfer to a standard six well culture plates, cells culturedin stromal medium attached to the plate in colony formation within 24hours (FIG. 2A). The vast majority of those cultured in induction mediumdid not attach at any point and remained as detached cell clusters(FIGS. 2B and 2C).

Example 4: Dithizone Staining—Zinc Concentration

The cells cultured in induction medium formed clusters that staineddithizone (DTZ), confirming zinc accumulation (FIGS. 3B-3C), and thosecultured in stromal medium did not stain with DTZ (FIG. 3A). As before,cells cultured in stromal medium attached to standard culture ware whilethose cultured in induction medium did not (FIG. 3A).

Example 5: Immunohistochemistry

The cells cultured in stromal medium lacked insulin expression, whilethe cells cultured in induction medium had strong insulin expression(FIG. 4).

Example 6: Glucose Challenge Assay

Male and female ASCs cultured in induction medium released insulin inresponse to glucose (FIG. 5). Insulin secretion was slightly higher(55.7 versus 62.7) at the higher glucose concentration. Insulinsecretion was significantly higher at high glucose concentrations (25and 55 mM) compared to a low glucose concentration (5.5 mM).

Example 7: Transmission Electron Microscopy—Ultrastructure

Cell ultrastructure was distinct between cells cultured in stromalversus induction medium. Notable differences were the presence abundantperinuclear mitochondria in the cells cultured in stromal medium (FIG.6A) and secretory granules in the induced cells (FIG. 6B).

Example 8: Electroimmunohistochemistry—Insulin Localization

Insulin labeling was localized to the cytoplasm of cells cultured ininduction medium, while there was no labeling in those cultured instromal medium (FIG. 7).

Example 9: Scanning Electron Microscopy—Surface Ultrastructure

Cells cultured in stromal medium tended to form loose, sphericalclusters while those in β cell induction medium formed larger, highlyorganized clusters with an irregular shape (FIG. 8). Proteinaceousmaterial was apparent on the surface of differentiated cell clusters(FIG. 8B).

Example 10: Gene Expression

This Example illustrates expression patterns of cells at various stagesof transdifferentiation.

Transcription Factor Expression

The mRNA levels of transcription factors (Nkx 6.1, Pax6, Isl1, andGlut-2) at Stage 1 tended to be lower than other induction stages (FIG.9). The mRNA levels of Pax6 and Glut2 in female samples had lowerexpression than male at induction stages 1 and 2 and induction stage 1,respectively (FIGS. 9B and 9D).

Pancreatic β-Cell Target Gene Expression

Insulin expression detectable only after stage 2 and 3 induction (FIG.10A). Glucagon expression was higher after stage 3 induction versus theother stages (FIG. 10B). Somatostatin levels were lower after stage 1induction compared to the others (FIG. 10C).

Oncogene Expression

There was no difference in ROS1, AKT1, RAB2A, or HK1 expression amonginduction stages (FIG. 11).

Insulin Expression

As shown in FIG. 12, pancreatic cells generated via the methodsdescribed herein (stages 1-3) express insulin. In particular, FIG. 12shows a Western blot demonstrating insulin protein produced by felinepancreatic beta cells generated from adipose derived stem cells. Columnsfrom left to right: Protein ladder, pancreatic beta cell cluster,pancreatic beta cell cluster, adipose derived stem cells cultured instromal medium, adipose derived stem cells cultured in stromal medium.

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All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The following statements are intended to describe and summarize variousembodiments of the invention according to the foregoing description inthe specification.

Statements:

-   -   1) A method comprising:        -   a. Stage 1: culturing adult adipose-derived multipotent            stromal (stem) cells (ASCs) for about 1-4 days in a first            culture medium comprising insulin-transferrin-selenium, a            TGFβ family member (e.g., activin A), an HDAC inhibitor            (e.g., sodium butyrate), and 2-mercapethanol to generate a            first population of cells that express at least one of the            following genes: Nkx 6.1, Pax6, Isl1, or Glut-2;        -   b. Stage 2: culturing the first population of cells for 4 to            8 days in a second culture medium comprising            insulin-transferrin-selenium, and taurine to generate a            second population of cells; and        -   c. Stage 3: culturing the second population of cells for 2            to 6 days in a third culture medium comprising            insulin-transferrin-selenium, taurine, glucagon-like peptide            1 (GLP-1), nicotinamide, pentagastrin, and betacellulin to            generate a third population of cells.    -   2) The method of statement 1, wherein the first culture medium,        the second culture medium, and the third culture medium further        comprise at least one, at least two, at least three, or at least        four of the following: basic fibroblast growth factor (bFGF),        hepatocyte growth factor (HGF), laminin, N-2 supplement, or B-27        supplement.    -   3) The method of statement 1 or 2, wherein the first culture        medium, the second culture medium, and the third culture medium        further comprise glucose, serum albumin (e.g., bovine serum        albumin).    -   4) The method of statement 1, 2, or 3, wherein the third culture        medium further comprises theophylline.    -   5) The method of statement 1-3, or 4, where the second        population of cells and/or the third population of cells express        insulin.    -   6) The method of statement 1-4 or 5, where cells in the first        population of cells do not express detectable amounts of insulin        mRNA as detected by quantitative polymerase chain polymerase        reaction.    -   7) The method of statement 1-5 or 6, further comprising        administering the third population of cells, or a portion        thereof, to a mammalian subject.    -   8) The method of statement 1-6 or 7, further comprising        administering the third population of cells, or a portion        thereof, to a feline subject.    -   9) The method of statement 1-7 or 8, wherein the adult        adipose-derived multipotent stromal (stem) cells (ASCs) are        cultured on an ultra-low attachment culture plate.    -   10) The method of statement 1-8 or 9, wherein cells in the first        population are cultured on an ultra-low attachment culture        plate.    -   11) The method of statement 1-9 or 10, wherein cells in the        first population are cultured on culture plates or in culture        vessels coated with protein.    -   12) The method of statement 1-10 or 11, wherein cells in the        second population expresses higher or lower levels of at least        one of the following genes: Nkx 6.1, Pax6, Isl1, or Glut-2 than        cells in the first population of cells.    -   13) The method of statement 1-11 or 12, further comprising        culturing cells from the third population and isolating insulin.    -   14) The method of statement 13, wherein the insulin is isolated        from the cultured cells.    -   15) The method of statement 13 or 14, wherein the insulin is        isolated from the culture medium.    -   16) A composition comprising a first population of cells made by        culturing adult adipose-derived multipotent stromal (stem) cells        (ASCs) from feline adipose tissue for about 1-4 days in a first        culture medium comprising insulin-transferrin-selenium, a TGFβ        family member (e.g., activin A), an HDAC inhibitor (e.g., sodium        butyrate), and 2-mercapethanol to generate a first population of        cells that express at least one of the following genes: Nkx 6.1,        Pax6, Isl1, or Glut-2.    -   17) The composition of statement 16, wherein the adult        adipose-derived multipotent stromal (stem) cells (ASCs) are        cultured on an ultra-low attachment culture plate.    -   18) A composition comprising a second population of cells made        by:        -   a. Stage 1: culturing adult adipose-derived multipotent            stromal (stem) cells (ASCs) from feline adipose tissue for            about 1-4 days in a first culture medium comprising            insulin-transferrin-selenium, a TGFβ family member (e.g.,            activin A), an HDAC inhibitor (e.g., sodium butyrate), and            2-mercapethanol to generate a first population of cells that            express at least one of the following genes: Nkx 6.1, Pax6,            Isl1, or Glut-2; and        -   b. Stage 2: culturing the first population of cells for 4 to            8 days in a second culture medium comprising            insulin-transferrin-selenium, and taurine to generate a            second population of cells.    -   19) The composition of statement 18, wherein the adult        adipose-derived multipotent stromal (stem) cells (ASCs) and/or        cells in the first population are cultured on an ultra-low        attachment culture plate.    -   20) The composition of statement 16-17 or 18, wherein cells in        the second population express higher or lower levels of at least        one of the following genes: Nkx 6.1, Pax6, Isl1, or Glut-2 than        cells in the first population of cells.    -   21) A composition comprising a third population of cells made        by:        -   a. Stage 1: culturing adult adipose-derived multipotent            stromal (stem) cells (ASCs) from feline adipose tissue for            about 1-4 days in a first culture medium comprising            insulin-transferrin-selenium, a TGFβ family member (e.g.,            activin A), an HDAC inhibitor (e.g., sodium butyrate), and            2-mercapethanol to generate a first population of cells that            express at least one of the following genes: Nkx 6.1, Pax6,            Isl1, or Glut-2;        -   b. Stage 2: culturing the first population of cells for 4 to            8 days in a second culture medium comprising            insulin-transferrin-selenium, and taurine to generate a            second population of cells; and        -   c. Stage 3: culturing the second population of cells for 2            to 6 days in a third culture medium comprising            insulin-transferrin-selenium, taurine, glucagon-like peptide            1 (GLP-1), nicotinamide, pentagastrin, and betacellulin to            generate a third population of cells.    -   22) The composition of statement 21, wherein the adult        adipose-derived multipotent stromal (stem) cells (ASCs), and/or        cells in the first population are cultured on an ultra-low        attachment culture plate.    -   23) A composition comprising at least four, or at least five, or        at least six, or at least seven, or at least eight, or at least        nine, or at least ten of the following: glucose, bovine serum        albumin, insulin-transferrin-selenium, a TGFβ family member        (e.g., activin A), an HDAC inhibitor (e.g., sodium butyrate),        2-mercapethanol, N-2 supplement, B-27 supplement, laminin,        mammalian hepatocyte growth factor (HGF), or basic fibroblast        growth factor (bFGF).    -   24) A composition comprising at least four, or at least five, or        at least six, or at least seven, or at least eight of the        following: glucose, bovine serum albumin,        insulin-transferrin-selenium, taurine, laminin, basic fibroblast        growth factor (bFGF), N-2 supplement, B-27 supplement, or        mammalian hepatocyte growth factor (HGF).    -   25) A composition comprising at least four, or at least five, or        at least six, or at least seven, or at least eight, or at least        nine, or at least ten, or at least eleven, or at least twelve of        the following: glucose, bovine serum albumin,        insulin-transferrin-selenium, taurine, glucagon-like peptide 1        (GLP-1), nicotinamide, non-essential amino acids, pentagastrin,        N-2 supplement, B-27 supplement, human hepatocyte growth factor        (HGF), basic fibroblast growth factor (bFGF), laminin, or        betacellulin.    -   26) The composition of statement 23, 24, or 25, which is        formulated as a culture medium.

The specific compositions, constructs, and methods described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention. Under no circumstances may the patent be interpreted to belimited to the specific examples or embodiments or methods specificallydisclosed herein. Under no circumstances may the patent be interpretedto be limited by any statement made by any Examiner or any otherofficial or employee of the Patent and Trademark Office unless suchstatement is specifically and without qualification or reservationexpressly adopted in a responsive writing by Applicants.

1. A method comprising: a. Stage 1: culturing adult felineadipose-derived multipotent stromal (stem) cells (ASCs) for about 1-4days in a first culture medium comprising at least four of thefollowing: glucose, bovine serum albumin, insulin-transferrin-selenium,activin A, sodium butyrate, 2-mercapethanol, N-2 supplement, B-27supplement, laminin, human hepatocyte growth factor (HGF), or basicfibroblast growth factor (bFGF) to generate a first population of cellsthat express at least one of Nkx 6.1, Pax6, Isl1, and Glut-2; b. Stage2: culturing the first population of cells in a second culture mediumcomprising at least four of the following: glucose, bovine serumalbumin, insulin-transferrin-selenium, taurine, laminin, basicfibroblast growth factor (bFGF), N-2 supplement, B-27 supplement, orhuman hepatocyte growth factor (HGF) to generate a second population ofcells; and c. Stage 3: culturing the second population of cells in athird culture medium comprising at least four of the following: glucose,bovine serum albumin, insulin-transferrin-selenium, taurine,glucagon-like peptide 1 (GLP-1), nicotinamide, non-essential aminoacids, pentagastrin, N-2 supplement, B-27 supplement, mammalianhepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF),laminin, or betacellulin to generate a third population comprising cellsthat can express insulin.
 2. The method of claim 1, further comprisingselecting one or more cells that express insulin.
 3. The method of claim1, further comprising selecting one or more cells that express insulinand separately expanding one or more of the selected cells that expressinsulin to generate a population of insulin-producing cells.
 4. Themethod of claim 1, further comprising administering cells from thesecond population to a mammal.
 5. The method of claim 1, furthercomprising administering cells from the third population to a mammal. 6.The method of claim 1 further comprising culturing one or more cellsfrom the third population and isolating insulin therefrom.
 7. Acomposition comprising the first population of cells made by the methodof claim 1, wherein the first culture medium comprisesinsulin-transferrin-selenium, activin A, sodium butyrate, and2-mercapethanol.
 8. The composition of claim 7, wherein cells in thefirst population of cells express Nkx 6.1, Pax6, Isl1, and Glut-2.
 9. Acomposition comprising the second population of cells made by the methodof claim 1, b. wherein the second culture medium comprisesinsulin-transferrin-selenium, and taurine.
 10. A composition comprisingthe third population of cells made by the method of claim 1, c. whereinthe third culture medium comprises insulin-transferrin-selenium,taurine, glucagon-like peptide 1 (GLP-1), nicotinamide, pentagastrin,and betacellulin.
 11. A composition comprising at least eight of thefollowing: glucose, bovine serum albumin, insulin-transferrin-selenium,activin A, sodium butyrate, 2-mercapethanol, N-2 supplement, B-27supplement, laminin, human hepatocyte growth factor (HGF), or basicfibroblast growth factor (bFGF).
 12. The composition of claim 11, whichis formulated as a culture medium.
 13. A composition comprising at leastseven of the following: glucose, bovine serum albumin,insulin-transferrin-selenium, taurine, laminin, basic fibroblast growthfactor (bFGF), N-2 supplement, B-27 supplement, or human hepatocytegrowth factor (HGF).
 14. The composition of claim 13, which isformulated as a culture medium.
 15. A composition comprising at leastten of the following: glucose, bovine serum albumin,insulin-transferrin-selenium, taurine, glucagon-like peptide 1 (GLP-1),nicotinamide, non-essential amino acids, pentagastrin, N-2 supplement,B-27 supplement, human hepatocyte growth factor (HGF), basic fibroblastgrowth factor (bFGF), laminin, or betacellulin.
 16. The composition ofclaim 15, which is formulated as a culture medium.